Reverse transcriptase (RT) is the crucial enzyme responsible for the synthesis of DNA from viral RNA for all retroviruses, including human immunodeficency virus (HIV) (Baltimore—70, Temin—70, Barre-Sinoussi et al—83). This process involves three different enzymatic activities: synthesis of the first DNA strand, degradation of the viral RNA strand in the DNA/RNA hybrid, and synthesis of the second DNA strand (for a review see Goff).
Retroviruses can exist either in exogenous or endogenous forms or both. The main difference is that an endogenous retrovirus has entered the germline and thus its DNA is present in all cells of the organism, in contrast to an exogenous retrovirus which is only able to enter cells with the appropriate receptor for that specific virus. Endogenous retroviruses (ERVs) in humans are called HERVs. The integrated proviral form of an endogenous retrovirus has the same fundamental structure as that of an exogenous retrovirus. The integration of some HERVs into the germline is thought to have occurred 35 to 45 million years ago. These retroviral sequences constitute 1% of the mammalian genome and hence also of the human genome. Thus, they are present in all human cells and inherited according to ordinary Mendelian laws.
Many families of HERVs are present in the human genome. The copy number per haploid human genome of HERVs varies between a single copy to thousands of copies for different families. The highest nucleotide sequence conservation is found in the pol gene. This feature has been used to derive relationship between HERV families. Although many of the HERVs and their ORFs are defective, mRNA transcripts have been detected from most HERV families in several tissue-types (reviewed in Wilkinson et al 1994, Leib-Mösch and Seifarth 1996). In addition, virion particles with polymerase and protease activity have been observed in normal human placentas, oocytes, teratocarcinomas, mammary carcinoma tissues and in salivary glands of some autoimmune patients (reviewed in Wilkinson et al. 1994, Urnovitz and Murphy 1996, Tönjes et al., 1997). Stress conditions like anoxia and UV irradiation might also induce the expression of HERVs as been observed for infectious retroviruses (reviewed by Duvic 1995).
Several HERVs have been localized to chromosomal breakpoints within the genome (Di Cristofano et al. 1995, Meese et al. 1996). Recombination between HERV sequences located at different chromosomal sites may cause genomic rearrangements including translocations, inversions, duplications and deletions. Such recombinatorial events are responsible for generating genomic instability that is an important feature of evolution. In addition, many tumors are characterised by specific genomic rearrangements thought to play a crucial role in the tumorogenesis. The relationship between presence of infectious retroviruses and development of tumors in the host suggests that HERVs are associated with cancer. Virion particles and retrovirus related antigens are frequently observed in primary tumor samples also in the absence of infectious viruses (Weiss 1984, Faff et al. 1992). In addition, antibodies directed to retroviral proteins have been demonstrated in the sera of patients with cancer (Weiss 1984). Moreover, HERV sequences are found to be highly expressed in certain tumor derived cell lines (reviewed in Löwer et al. (1996).
Endogenous proviruses can also recombine with exogenous variants (Martinelli et al. 1990). Novel recombinants receive an altered phenotype. This might contribute to the rapid evolution of new infectious retroviruses. The env gene is mainly affected since mutations in other parts may be deleterious and not compatible with particle formation. Some retroviral strains are found to be symbiotic in one species and pathogenic in another. These findings indicate a severe problem in xenotransplantations. Symbiotic ERVs provided by the transplanted tissues could be pathogenic in the new host. They might be able to interact with cell surface receptors needed for retroviral fusion present on cells of the new host. In addition, recombination with endogenous retroviral sequences within the genome of the tissue recipient can occur and create new infectious retroviruses that might spread in the population. Both endogenous and exogenous retroviruses can contribute to vertical and horizontal transmission of genetic material within and between species and provide mechanisms for evolution of new pathogenic agents.
Both infectious retroviruses and ERVs have been found to exhibit immuno regulatory functions (reviewed in Krieg et al. 1992). These effects have mainly been studied in mice but there are some observations that support existence of similar mechanisms for HERVs. If HERV sequences can deregulate an immune response they can cause autoimmune diseases. These effects could be directly modulated by HERV proteins or indirectly by influencing the expression of molecules involved in the immune response. HERV proteins can be exposed on the cell surface. Loss of self tolerance towards these protein sequences could cause autoimmune reactions against cells exposing them. Antibodies produced following retroviral infection might be crossreactive with HERV encoded proteins and responsible for loss of tolerance. This phenomenon has been observed in transgenic mice (Zinkernagel et al. 1990). In addition, loss of tolerance to ERV encoded env proteins was observed in mice that spontaneously developed an autoimmune glomerulonephritis (reviewed in Krieg et al. 1990 and 1992). Some observations in humans might support existence of crossreactive antibodies between endogenous and exogenous retroviral proteins. Antibodies against retroviral proteins have been detected in sera of autoimmune patients (reviewed in Krieg et al. 1992, Urnovitz and Murphy 1996). In certain cases, the sera were found to react also with exogenous retroviruses. Retroviral infections have been associated with the onset of autoimmune diseases (reviewed in Krieg et al. 1990 and 1992). Moreover, infectious retroviral proteins show partial similarity to self antigens that are frequent targets for autoantibodies (reviewed in Krieg et a 1992). They could trigger autoimmune responses towards such targets, a mechanism referred to as molecular mimicry. However, similarities of HERV proteins to known self antigens have not yet been detected.
Assays for RT activity have become accepted techniques for the detection and quantification of retroviruses in cell cultures. They are together with p24 antigen assays used as confirmatory tests for HIV isolation (Jackson—88, Gupta—87). RT is also one of the main targets in the attempt to find efficient antivirals against HIV. A conventional RT activity assay is performed by utilizing a soluble enzyme assay with an artificial template-primer construction and tritiated deoxynucleotide triphosphate as nucleotide substrate (Baltimore-71, Lee et al—87). This early system was based on detection of incorporation of radioactivity into trichloroacetic-acid (TCA) precipitable RNA/DNA hybrids. Use of beta emitting nucleotides requires the use of scintillation fluids for detection of radioactivity, which often results in poor reproducibility due to quenching problems. This method is relatively cumbersome and not easily adapted to large scale screening of large numbers of samples. It is also very sensitive to the effects of disturbing factors in the samples.
During the last decade of intensive research on HIV, the RT assay has been improved using different techniques. The introduction of I125 labeled substrate gave an increased sensitivity and eliminated quenching and the use of scintillation fluids (Neumüller et al—90). The introduction of templates or primers linked to solid phases simplified the separation between substrate and product, eliminated the need of TCA precipitations and resulted in a “one tube RT assay” (Gronowitz et al—90, EP 0 447 442 B1).
More recently the use of radioactivity as label in RT assay has been eliminated by the use of modified nucleotide bases containing either antigenic eptiopes or structures with high affinity to defined ligands. The presence of these epitopes or structures in the newly synthesized RNA/DNA hybrid is then used for binding of antibodies or ligands conjugated with e.g. ELISA enzymes. The amount of ELISA enzymes bound is then determined in a secondary enzyme assay.
Porstmann et al 1991 utilizes 5-bromo-deoxyuridine (BrdU) triphosphate as nucleotide substrate in RT assay. The amount of BrdUMP incorporated is in a secondary step determined in an immunoassay using alkaline phosphatase conjugated monoclonal anti-BrdU antibodies.
Eberle and R. Scibl 1992 measure the incorporation of digoxigenin labeled dUTP into newly synthesized DNA instead of radioactively labeled dTTP. To be able to perform the separation of non-incorporated nucleotides from the newly synthesized DNA, biotin labeled dUTP is also added to the reaction mixture. After reverse transcription, the newly synthesized double labeled DNA is immobilized on streptavidin coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies. This procedure has been the base for a kit RT-assay which is commercially available from Boehringer Mannheim.
Urabe et al 1994 has developed a non-radioactive RT-assay based on incorporation of biotin-dUTP in an immobilized odT/prA construct. The amount of incorporated nucleotide substrate is measured photometrically after addition of streptavidin conjugated alkaline phosphatase. Closest prior aft to the current invention was developed by Ekstrand et al 1996. In that assay poly (rA) covalently bound to the wells of a 96 well microtiter plate serves as template for the incorporation of BrdUMP during the reverse transcription step. The amount of BrdUMP incorporated into DNA is then determined immunologically according to a similar procedure as used by Porstmann et al 1991. The method is available as RT determination kits from Cavidi Tech, Uppsala, Sweden.
Another principle for detection of RT-activity, commercially exploited by NEN (New England Nuclear, USA), is utilization of sequence specific probes for detection of newly synthesized cDNA. The enzymatic reaction utilizes a heteropolymeric RNA molecule with a 20-base oligonucleotide primer complementary to the RNA sequences near the 5′-end. A complete cDNA strand is produced during the RT-reaction. After hydrolysis of the template RNA the cDNA is hybridized with two different oligonucleotide probes, the capture and the detection probe. The capture probe is used for binding the cDNA to a microplate well. The detection probe is conjugated to horseradish peroxidase, which after washing to remove unused nucleotide substrate and free probes gives a color reaction.
The detection sensitivity in the last type of assay can be increased by polymerase chain reaction amplification of the cDNA produced by the RT reaction. The amplified DNA can thereafter be detected with different types of labeled probes (Silver 93, Heneine 1995, U.S. Pat. Nos. 5,817,457, 5,849,494).
For some applications of the knowledge of RT activity in biological samples it is desirable to use a RT assay giving quantitative results. Such applications comprise disease or disorder monitoring where RT assay results from measurements made at different times are to compared, and diagnosing of diseases or disorders where the level of RT activity compared to standard levels indicates if the patient is at risk or in deed is suffering from the disease or disorder in question.
In some instances it is desirable to use an assay which is as sensitive as possible, i.e. is able to measure as small amounts of RT activity as possible, in biological samples, so that the presence and magnitude of RT activity can be related to disorders and diseases at an early stage.